1. Technical Field
The present invention relates to optoelectronic control of solid-state nanopores and nanopore arrays, and applications thereof.
2. Discussion of Related Art
Nanopores, pores of nanometer dimensions in an electrically insulating membrane, have shown promise for use in a variety of sensing applications, including single molecule detectors. The nanopores used in such applications can be biological protein channels in a lipid bilayer or a pore in a solid-state membrane. Solid-state nanopores are generally made in silicon compound membranes, one of the most common being silicon nitride. Solid-state nanopores can be manufactured with several techniques including ion-beam sculpting of silicon nitride and using e-beam lithography.
The use of nanopores in single-molecule detection employs a detection principle based on monitoring the ionic current of an electrolyte solution passing through the nanopore as a voltage is applied across the membrane. When the nanopore is of molecular dimensions, passage of molecules causes interruptions in the open pore current level. The temporal variation in current levels leads to a translocation event pulse. These detection methods are described at length in: Kasianowicz JJ, Brandin E, Branton D, Deamer D W, (1996) “Characterization of individual polynucleotide molecules using a membrane channel.” Proc Nat Acad Sci 93:13770-13773; Akeson, M, Branton, D, Kasianowicz J, Brandin E, and Deamer D, (1999) Biophys. J. 77: 3227-3233; Meller A, Nivon L, Brandin E, Golovchenko J, Branton D, (2000) Proc Nat Acad Sci 97: 1079-1084, all of which are herein incorporated by reference in their entireties.
Nanopore detection techniques have been used for biomolecule detection. For example, various nanopore sequencing methods have been proposed. In 1994, Bezrukov, Vodyanoy and Parsegian showed that one can use a biological nanopore as a Coulter counter to count individual molecules (Counting polymers moving through a single ion channel, Nature 370, 279-281 (1994) incorporated herein by reference). In 1996, Kasianowicz, Brandin, Branton and Deamer proposed an ambitious idea for ultrafast single-molecule sequencing of single-stranded DNA molecules using nanopore ionic conductance as a sensing mechanism (Characterization of individual polynucleotide molecules using a membrane channel, Proc. Nat. Acad. Sci. USA 93 13770-13773 (1996), incorporated herein by reference).
The methods seek to effectively determine the order in which nucleotides occur on a DNA (or RNA) strand. The theory behind nanopore sequencing concerns observed behavior when the nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. Under these conditions an electrical current that results from the conduction of ions through the nanopore can be observed. The amount of current which flows is sensitive to the size of the nanopore. When a biomolecule passes through the nanopore, it will typically create a change in the magnitude of the current flowing through the nanopore. Electronic sensing techniques are used to detect the ion current variations, thereby sensing the presence of the biomolecules.
U.S. Pat. No. 6,428,959, the entire contents of which are herein incorporated by reference, describes methods for determining the presence of double-stranded nucleic acids in a sample. In the methods described, nucleic acids present in a fluid sample are translocated through a nanopore, e.g., by application of an electric field to the fluid sample. The current amplitude through the nanopore is monitored during the translocation process and changes in the amplitude are related to the passage of single- or double-stranded molecules through the nanopore. Those methods find use in a variety of applications in which the detection of the presence of double-stranded nucleic acids in a sample is desired.
There are numerous challenges to develop effective nanopore detection techniques. Two of the most significant challenges facing nanopore-assisted genomic sequencing are that molecular passage across the nanopore is higher than desired, and current throughput is lower than desired due to nanopores consistently getting permanently blocked. It would be desirable to provide solid-state nanopores with surface characteristics that can be selectively modified to modulate translocation speed and unblock clogged pores.